Cytocidal Actions of Parasporin-2, an Anti-tumor Crystal Toxin from Bacillus thuringiensis

Abstract

Parasporin-2, a new crystal protein derived from noninsecticidal and nonhemolytic Bacillus thuringiensis, recognizes and kills human liver and colon cancer cells as well as some classes of human cultured cells. Here we report that a potent proteinase K-resistant parasporin-2 toxin shows specific binding to and a variety of cytocidal effects against human hepatocyte cancer cells. Cleavage of the N-terminal region of parasporin-2 was essential for the toxin activity, whereas C-terminal digestion was required for rapid cell injury. Protease-activated parasporin-2 induced remarkable morphological alterations, cell blebbing, cytoskeletal alterations, and mitochondrial and endoplasmic reticulum fragmentation. The plasma membrane permeability was increased immediately after the toxin treatment and most of the cytoplasmic proteins leaked from the cells, whereas mitochondrial and endoplasmic reticulum proteins remained in the intoxicated cells. Parasporin-2 selectively bound to cancer cells in slices of liver tumor tissues and susceptible human cultured cells and became localized in the plasma membrane until the cells were damaged. Thus, parasporin-2 acts as a cytolysin that permeabilizes the plasma membrane with target cell specificity and subsequently induces cell decay.

Overexpression and Cytocidal Activity of Parasporin-1 from Bacillus thuringiensis against Human Cancer Cell Lines

“…Interestingly, a goldfish (Carassius auratus) cell line, GF-Scale, is also not sensitive to the three crude preparations of parasporal proteins containing PS1Aa1, PS2Aa1, and PS3Aa1, respectively [21,22]. The cytotoxicity spectra of the four PS proteins summarize the results obtained from recent investigations [23][24][25][26]. It involves 13 human cell lines originating from various tissues, nine from tumors, four non-cancer normal tissues and five non-human cells (two monkey and three rodent cell lines).…”

Crystal Structure of the Parasporin-2 Bacillus thuringiensis Toxin That Recognizes Cancer Cells

“…Limited proteolysis of the nascent protoxin with proteinase K removes 51 and 36 residues from its N-terminus and C-terminus, respectively, to produce a 30-kDa active form that has strong cytocidal activity against various human cells. 12,16 It is highly toxic to cells of the hepatocellular carcinoma cell line HepG2 and to CACO-2 colon cancer cells, as well as to human leukemia T-cells, including MOLT-4, Jurkat, and HL-60 cells; the 50% lethal doses (LD 50 ) for these cells are all around 20 ng/ml. 16 It is less toxic (LD 50 N 1 μg/ml) to normal HC hepatocytes and HeLa cervical cancer cells.…”

Parasporin-1, a Novel Cytotoxic Protein from Bacillus thuringiensis, Induces Ca2+ Influx and a Sustained Elevation of the Cytoplasmic Ca2+ Concentration in Toxin-sensitive Cells

“…10) from the A1547 strain of B. thuringiensis (6). In addition to the different cytotoxic spectra of parasporin-1 and parasporin-2, parasporin-2 reduced the membrane potential of treated cells at the same concentration used for the cytotoxic assay (45), indicating that parasporin-1 and parasporin-2 exert their cytotoxic effects on mammalian cells by different mechanisms. Cleavage of procaspase-3 and PARP was assessed by Western blot analysis using an anticaspase-3 and anti-PARP monoclonal antibodies, respectively.…”